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J Bacteriol. Feb 2007; 189(3): 958–967.
Published online Dec 1, 2006. doi:  10.1128/JB.01474-06
PMCID: PMC1797333

Expression of the Major Porin Gene mspA Is Regulated in Mycobacterium smegmatis[down-pointing small open triangle]


MspA is the major porin of Mycobacterium smegmatis and is important for diffusion of small and hydrophilic solutes across its unique outer membrane. The start point of transcription of the mspA gene was mapped by primer extension and S1 nuclease experiments. The main promoter driving transcription of mspA was identified by single point mutations in lacZ fusions and resembled σA promoters of M. smegmatis. However, a 500-bp upstream fragment including PmspA in a transcriptional fusion with lacZ yielded only low β-galactosidase activity, whereas activity increased 12-fold with a 700-bp fragment. Activation of PmspA by the 200-bp element was almost eliminated by increasing the distance by 14 bp, indicating binding of an activator protein. The chromosomal mspA transcript had a size of 900 bases and was very stable with a half-life of 6 minutes, whereas the stabilities of episomal mspA transcripts with three other 5′ untranslated region (UTRs) were three- to sixfold reduced, indicating a stabilizing role of the native 5′ UTR of mspA. Northern blot experiments revealed that the amount of mspA mRNA was increased under nitrogen limitation but reduced under carbon and phosphate limitation at 42°C in stationary phase in the presence of 0.5 M sodium chloride, 18 mM hydrogen peroxide, and 10% ethanol and at acidic pH. These results show for the first time that M. smegmatis regulates porin gene expression to optimize uptake of certain nutrients and to protect itself from toxic solutes.

The fast-growing Mycobacterium smegmatis is nonpathogenic and dwells in the soil, where it faces quickly changing environmental conditions (14, 34). One of the most striking features of mycobacteria is their unique cell wall, which according to several models comprises a supported outer membrane (OM) of very low fluidity and extremely low permeability to both hydrophobic and hydrophilic solutes (4, 25, 31). The sturdiness of the cell wall and the impermeable OM protects mycobacteria from environmental stresses and contributes to their intrinsic resistance to many antibiotics (4). However, despite this apparent emphasis on the protective function of the OM, mycobacteria must be able to acquire nutrients and to release waste products. Porins are water-filled protein channels that span the OM of mycobacteria and enable the diffusion of small and hydrophilic solutes (28). MspA was identified as a channel-forming protein in chloroform-methanol extracts of M. smegmatis (29). The OM permeability of an M. smegmatis ΔmspA mutant for cephaloridine and glucose was reduced nine- and fourfold, respectively (42). The number of pores in the cell wall of M. smegmatis dropped from 2,400 to 800 per cell in the mspA mutant in the exponential growth phase as shown by electron microscopy (9, 43). These results showed that MspA is the major porin out of four Msp porins of M. smegmatis. Deletion of the mspA, mspC, and mspD genes in a triple mutant strongly reduced the growth rate of M. smegmatis, underlining the importance of the Msp porins for nutrient uptake (43). MspA is the first mycobacterial OM protein whose structure has been resolved at the atomic level. The crystal structure illustrated that eight MspA monomers constitute a single channel of 9.6 nm in length (10). This protein architecture is completely different from that of the trimeric porins of gram-negative bacteria, which have one pore per monomer and are approximately 4 nm long (17). These results established MspA as the first member of a new class of porins (28).

Regulation of porin gene expression and activity is of utmost importance for Escherichia coli and other gram-negative bacteria in order to adapt to environmental changes both inside and outside a mammalian host. Multiple factors, such as osmolarity, pH, growth phase, temperature, oxidative stress, or the presence of toxic compounds, alter the expression of genes encoding the general diffusion porins OmpF and OmpC at the transcriptional level by the two-component regulatory system EnvZ-OmpR (19, 33). In many cases, the total number of porins is maintained, but the ratio of the larger pore OmpF to the smaller pore OmpC is dependent on the level of phosphorylated OmpR (33). In addition, other proteins, such as RpoS, AckA, catabolite activator protein, H-NS, and integration host factor, regulate transcription of porin genes in E. coli in response to nutrient limitation, growth phase, and osmolarity of the environment (19). Recently, the two-component stress response system CpxA-CpxR (3) and a novel putative RNA regulator named ipeX (5) have been discovered to play a role in regulation of ompF and ompC. Both ompF and ompC are repressed by the micF and micC antisense RNAs, respectively, and solely ompC is repressed by RseX. These RNAs are complementary to the corresponding 5′ untranslated region (UTRs), inhibit ribosome assembly, and induce mRNA degradation upon binding to their cognate mRNAs (6-8). Transcription of micF and micC integrates signals from multiple regulatory pathways (6, 7).

Considering the importance of controlling the OM permeability and the extensive and complex regulation of porin gene expression in gram-negative bacteria, it is surprising that nothing is known about the regulation of expression of porin genes in mycobacteria. In this study, we have chosen the major porin gene mspA of M. smegmatis to examine whether and how porin genes in mycobacteria are regulated. We have identified the major promoter of mspA and multiple signals that drastically alter the amount of mspA mRNA. Further, the 5′ UTR of mspA appears to play an important role in stabilizing the mspA transcript and preventing mRNA decay.


Bacterial strains and growth conditions.

Mycobacterium smegmatis SMR5 (38) was routinely grown at 37°C with shaking in Middlebrook 7H9 liquid medium (Difco Laboratories) supplemented with 0.2% glycerol and 0.05% Tween 80 or on Middlebrook 7H10 agar (Difco Laboratories) supplemented with 0.2% glycerol unless indicated otherwise. Escherichia coli DH5α was used for all cloning experiments and was routinely grown in LB medium at 37°C. Hygromycin was used at concentrations of 200 μg ml−1 for E. coli and 50 μg ml−1 for M. smegmatis.

Identification of growth conditions that alter transcription of the mspA gene of M. smegmatis.

Cultures of M. smegmatis SMR5 for RNA preparation were grown to an optical density at 600 nm (OD600) of 0.8 to 1.0 at 37°C in Hartmans-de Bont (HdB) medium (41). HdB medium was modified by changing the amounts of ingredients when necessary for the individual experiments. For the pH experiments, citrate buffer (0.75 M sodium citrate, 0.75 M citric acid) was included in HdB medium to a final concentration of 50 mM, and the appropriate pH was adjusted with 1 M NaOH or 1 M HCl. Cells were harvested by centrifugation and resuspended in equal volumes of the growth medium. Cultures were then further incubated for 3 h at 37°C unless otherwise indicated.

RNA preparation.

After incubation at the above mentioned conditions, cultures were mixed with half of the volume of precooled killing buffer (20 mM Tris-HCl, 5 mM MgCl2, 20 mM NaN3). The cell suspension was incubated on ice for 5 min. Cells were harvested by centrifugation, resuspended in RA1 buffer with β-mercaptoethanol (Nucleospin RNAII kit), and lysed by agitation with glass beads (FastRNA Tubes-Blue) in a FastPrep FP120 bead beater apparatus (Bio-101) by three 20-second pulses at level 6.5. Suspensions were cooled on ice for 5 min between agitation steps. Further processing of the sample to purify the RNA was performed using the Nucleospin RNAII kit (Macherey-Nagel) following the manufacturer's instructions.

Northern blot analysis.

One to three micrograms of total RNA from M. smegmatis was loaded on a denaturing RNA gel (1.2% agarose, 1× morpholinepropanesulfonic acid [MOPS] [1× MOPS is 200 mM MOPS, 50 mM sodium acetate, and 10 mM EDTA, pH 7], 3.15% formaldehyde). Gel electrophoresis was done at 70 V for 3 to 4 h in 1× MOPS. The RNA was transferred to a positively charged nylon membrane by using a vacuum blot apparatus at 6,000 Pa and by adding denaturing buffer (50 mM NaOH, 10 mM NaCl) and neutralizing buffer (0.1 M Tris HCl, pH 7.4) for 10 min and 20× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate, pH 7) for 3 h. After the transfer, the RNA was cross-linked twice to the membrane with UV light at 1,200 kJ. The membrane was incubated at 68°C in a hybridization oven with prehybridization solution (50% formamide, 5× SSC, 2× blocking reagent, 0.1% N-laurylsarcosine, 0.02% sodium dodecyl sulfate [SDS]) for 2 h and overnight at 68°C with an RNA probe. The membrane was washed twice for 5 min each time in 2× SSC-0.1% SDS at room temperature and three times for 15 min each time in 0.2× SSC-0.1% SDS at 68°C. For detection, the membrane was equilibrated in 1× maleic acid buffer (1 M maleic acid, 0.3 M Na citrate, pH 7.5) for 1 minute, then in blocking buffer (1× blocking reagent in 1× maleic acid buffer) for 30 min, and finally in blocking buffer with alkaline phosphatase-conjugated antibody against digoxigenin for 30 min. Then, the membrane was washed twice for 15 min each time in 1× maleic acid buffer and for 5 min in detection buffer (0.1 M diethanolamine). All incubation steps were performed with gentle shaking at room temperature. The membrane was completely covered with CDP* (DIG Northern starter kit; Roche) and incubated in darkness for 5 min. Chemiluminescent signals on the membrane were photographed, and detected bands were densitometrically measured and quantified in a UVP EpiChem3 Darkroom using LabWorks software. Detection of the 16S rRNA was performed as a control, and the amounts of mspA transcripts were normalized to that of 16S rRNA in the same sample.

Dot blot analysis.

One to three micrograms of total RNA from M. smegmatis was loaded directly on a positively charged nylon membrane. To avoid differences in dot size when using the same amounts of total RNA, the RNA concentrations of all samples were adjusted to 500 to 600 ng μl−1. The membrane was dried and cross-linked as described above. Hybridization and detection were performed by methods similar to those of Northern blot analysis.

RNA probe construction.

Probes were amplified from chromosomal DNA from M. smegmatis SMR5 by PCR using specific primers (see Table S4 in the supplemental material) for the sigA gene and the 16S rRNA gene. Probes for the mspA and lacZ genes were amplified from the plasmids pPOR6 and pMlacZsd (see Table S1 in the supplemental material), respectively. A recognition site for T7 RNA polymerase was added to the 5′ ends of the reverse primers. The PCR products were purified by elution from agarose gels and quantified. Two hundred nanograms was used for in vitro transcription using the Roche DIG RNA labeling mix according to the manufacturer's instructions. The RNA probes were purified by elution from agarose gels, precipitated, and resuspended in 50 μl of RNase-free water. For hybridization overnight, 10 μl of an RNA probe was added to 10 ml prehybridization solution.

RNA stability and half-life.

Cultures of M. smegmatis were grown to an OD600 of 0.8 to 1.0, and then 10 ml of the cell suspension was used for RNA preparation as described above for time zero. Simultaneously, rifampin was added to the incubating cultures to a final concentration of 200 μg ml−1 to inhibit further transcription. Another 10 ml of each culture was used for RNA preparation 1, 2, 3, and 5 min after the addition of rifampin to monitor time-dependent RNA decay with Northern hybridization (see above).

Primer extension experiments.

For primer extensions, the mspA gene-specific reverse primer MP-PE2 was designed (see Table S2 in the supplemental material; its position is depicted in Fig. Fig.2).2). End-labeled primer (labeled with γ-32P) (0.4 pmol) was annealed to 10 μg total RNA from exponential-phase cultures of M. smegmatis SMR5 in 1× hybridization buffer (1× hybridization buffer is 0.15 M KCl, 0.01 M Tris HCl, pH 8, and 1 mM EDTA). Samples were denatured for 5 min at 95°C and hybridized for 2 h at 50°C. cDNA was synthesized using avian myeloblastosis virus reverse transcriptase and the corresponding reverse transcription buffer (250 mM Tris HCl, pH 8, 130 mM MgCl2, 75 mM dithiothreitol, 2 mM each deoxynucleoside triphosphate). The reaction mixture was incubated at 42°C for 1 h.

FIG. 2.
Upstream region of the mspA gene. Genes with annotations are msmeg0951 (transcriptional regulator of the TetR family), msmg0958 (cytochrome P450), and hemL (glutamate-1-semialdehyde-2,1-aminomutase). Other genes represent open reading frames of unknown ...

Five micrograms of plasmid DNA from pPOR6 (see Table S1 in the supplemental material) and the primer MP-PE2 were used for sequencing mspA. Plasmid DNA was denatured in 0.2 M NaOH and 0.2 mM EDTA for 30 min at 37°C, precipitated, and redissolved in 7 μl H2O. The end-labeled MP-PE2 (labeled with γ-32P) was annealed, and sequencing was performed using the USB Sequenase version 2.0 sequencing kit following the manufacturer's guidelines. The sequencing reactions together with the corresponding primer extension reactions were analyzed on a 8% denaturing polyacrylamide gel (7 M urea).

Nuclease S1 mapping.

RNA from M. smegmatis cultures during exponential growth phase was prepared as described above. The primer MP-P2 (see Table S2 in the supplemental material) was radioactively labeled (see “Primer extension experiments” above), precipitated, and resuspended in 10 μl TE (Tris-EDTA) buffer. PCR was performed using 2 μg of StyI-linearized plasmid pPOR6 (see Table S1 in the supplemental material) and 2 pmol of the labeled primer MP-P2, which yielded a radioactive DNA probe of 320 bp. The PCR product was purified by elution from a 6% polyacrylamide gel (7 M urea). The probe was precipitated together with 40 μg RNA from M. smegmatis and resuspended in 40 μl hybridization buffer [40 mM piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES), pH 6.4, 400 mM NaCl, 1 mM EDTA, 80% formamide]. Hybridization was carried out at 50°C for 90 min, at 45°C for 90 min, and at 40°C for 60 min. Nonhybridized RNA was digested by the addition of 30 U nuclease S1 and 300 μl S1 reaction buffer (50 mM sodium acetate, pH 4.6, 28 mM NaCl, 4.5 mM ZnSO4, 20 μg/ml single-stranded DNA) and incubated at 37°C for 1 h. After the addition of 50 μl stop solution (4 M NH4 acetate, 0.1 M EDTA), the sample was precipitated with ethanol, resuspended in 3 μl TE buffer with 0.4 μg/ml RNase A, mixed with 1 μl denaturing loading buffer, and run on a 0.1-mm-thick 8% denaturing polyacrylamide gel (7 M urea).

Construction of β-galactosidase-expressing vectors.

To reduce background activity, the transcriptional terminator of gene 32 of phage T4, ttT4g32, which has been shown to efficiently terminate transcription in mycobacteria (45), was cloned upstream of the lacZ gene. Therefore, primers T4g32T_Pme_Sph_fwd and T4g32T_Pme_Sph_rev were used (see Table S2 in the supplemental material). They were phosphorylated using T4 polynucleotide kinase (New England BioLabs) following the recommendations of the manufacturer. Five hundred picomoles of each phosphorylated primer was mixed and annealed by heating to 90°C and cooling down to 65°C over 20 min. Hybridization resulted in a ttT4g32 fragment, which was inserted via restriction sites for PmeI and SphI into the pMlacZsd plasmid (kindly provided by Sabine Ehrt, Cornell University, NY) to yield pML163.

To obtain vectors harboring transcriptional fusions of the mspA promoter fragments to the lacZ gene, the mspA promoter fragments were inserted upstream of the lacZ gene into pML163 via the PmeI and SphI restriction sites. The mspA promoter fragments were amplified by PCR from the template plasmid pPOR6 (29) using the following primers: mpp4 and mpp1-fwd for PmspA500 bp (resulting in pML164), mpp4 and mpp10 for PmspA600 bp (pML808), mpp4 and mpp11 for PmspA700 bp (pML809), mpp4 and mpp12 for PmspA800 bp (pML810), mpp4 and mpp13 for PmspA900 bp (pML811), mpp4 and mpp14 for PmspA1000 bp (pML812), mpp4 and mpp7 for PmspA1100 bp (pML167), and mpp7 and mpp9 for PmspA600 bp up (pML801). To obtain the plasmids with constant 500-bp proximal fragments but different 200-bp fragments distal to mspA, pML164 was digested with PmeI and ligated with similarly cut PCR fragments obtained with oligonucleotide pairs mpp15/mpp16 (pML823; 200 bp of −500 to −700 relative to mspA), mpp17/mpp18 (pML824; 200 bp of −700 to −900), and mpp19/mpp20 (pML825; 200 bp of −900 to −1100). The entire mspA promoter fragments were sequenced to ensure the right orientation and the absence of secondary site mutations. The verified plasmids were electroporated in M. smegmatis SMR5.

Mutation of the mspA promoter.

Promoter point mutations in the PmspA1100 bp fragment were obtained by the combined chain reaction as described earlier (21) using the pPOR6 vector as the template. The end primers were mpp4 and mpp7, and the mutation primers were PromT1C, PromA2C, and PromT6C (see Table S2 in the supplemental material). The mutation primers were phosphorylated using T4 polynucleotide kinase (New England BioLabs) following the manufacturer's manual. The resulting plasmids had the following mutations: T to C at position −147 (pML820), A to C at −146 (pML821), and T to C at −142 (pML822) relative to the mspA start codon. The entire mspA promoter fragments were sequenced to verify the mutations and to ensure the absence of secondary site mutations. Verified plasmids were electroporated in M. smegmatis SMR5.

β-Galactosidase activity measurements.

To determine β-galactosidase activity of recombinant M. smegmatis, cells were grown in Middlebrook 7H9 to an OD600 of 0.8 to 1. For measuring activity at different pH, cells were grown in HdB medium as described above. After incubation at different conditions for 3 h, 1-ml samples were taken, harvested, and resuspended in the same volume of neutral HdB medium. OD600 was determined with 100-μl portions of these samples. The remaining 900 μl was sonicated using a Misonix sonicator 3000 with the following settings: two complete intervals of 20 seconds total pulse time consisting of pulses for 0.9 s and breaks for 0.5 s at strength 3. Between intervals, the samples were kept on ice. Two hundred microliters of sonicated cells was mixed with 800 μl of freshly prepared LacZ medium consisting of 1× LacZ buffer (1× LacZ buffer is 60 mM Na2HPO4, 40 mM NaH2PO4, 3 mM MgCl2, 10 mM KCl, and 50 mM β-mercaptoethanol) and 600 μl of Middlebrook 7H9 or neutral HdB medium, depending on the growth medium. The samples were prewarmed at 28°C for 15 min, and 200 μl o-nitrophenyl-β-d-galactopyranoside (ONPG) (4 mg ml−1 in 1× LacZ buffer) was added. Samples were incubated at 28°C until they turned yellow. Then, β-galactosidase was inactivated by adding 500 μl 1 M Na2CO3, and the time was determined. Absorption was measured at 420 and 550 nm, and Miller units were calculated.


Identification of the mspA promoter.

In a first step to identify the promoter of the mspA gene, the transcriptional start point (TSP) of mspA was determined. To this end, M. smegmatis was grown to the exponential growth phase and RNA was prepared. Primer extension experiments revealed a strong signal for mspA transcripts starting with a G at position −135 relative to the start codon (Fig. (Fig.1A).1A). S1 nuclease mapping was performed with the same RNA and confirmed this TSP (Fig. (Fig.1B).1B). A much weaker signal corresponding to longer transcripts with a TSP at G −153 bp upstream of mspA was also observed (Fig. (Fig.1B).1B). A search within the 5′ upstream region (5′ UTR) of the mspA gene for sequences with similarities to the consensus sequence for promoters in M. smegmatis (TATAAT with 100% conservation for T, 93% for A, 50% for T, 57% for A, 43% for A, and 71% for T [26]) revealed a potential promoter sequence TATGTT upstream of the main TSP at position −135 (Fig. (Fig.2).2). To examine whether the sequence TATGTT at position −147 represents the mspA promoter, a transcriptional fusion of a 1,100-bp fragment upstream of mspA to the E. coli lacZ gene was constructed (pML167 [see Table S1 in the supplemental material]). Then, point mutations at the most highly conserved positions, i.e., positions 1, 2, and 6, were introduced into the putative promoter sequence TATGTT within the complete 1,100-bp fragment upstream of mspA. The plasmids with fusions of the wild-type and mutated mspA promoter fragments to lacZ were transformed into M. smegmatis, and the β-galactosidase activities of these strains were measured (Fig. (Fig.3).3). The A2C mutation completely eliminated β-galactosidase activity. The T1C and T6C mutations reduced β-galactosidase activity more than 30-fold. These results demonstrated that the sequence TATGTT at position −147 is indeed the sole promoter for initiation of transcription of the mspA gene under these conditions.

FIG. 1.
Start points of transcription of the mspA gene and dependence of mspA mRNA levels on growth phase. (A) The primer extension experiment was performed using RNA from M. smegmatis SMR5 and the primer MP-PE2 labeled by phosphorylation of the 5′-OH ...
FIG. 3.
Mutational analysis of the mspA promoter. The activity of β-galactosidase of M. smegmatis SMR5 with plasmids harboring fusions of lacZ with different fragments upstream of mspA was measured and is indicated in Miller units (MU). Mutational analysis ...

A very long 5′ upstream activating region is required for full activity of the mspA promoter.

Since most bacterial promoters are located within 200 bp upstream of the gene of interest (26), a 500-bp fragment upstream of the start codon of the mspA gene was initially fused to the lacZ gene to examine regulation of the mspA promoter. However, transcription from the mspA promoter in this fragment was very low compared to other mycobacterial promoters (not shown). To test whether this fragment may not contain all functions for full promoter activity, longer fragments of the DNA upstream of mspA were fused to lacZ. The β-galactosidase activity rose by 7- and 12-fold as a result of increasing the length of the promoter fragment to 600 bp and 700 bp, respectively. This activity did not increase further with longer fragments (Fig. (Fig.4A4A).

FIG. 4.
Identification of a 5′-upstream activating region of the mspA promoter. The activity of β-galactosidase of M. smegmatis SMR5 carrying plasmids harboring fusions of lacZ with different fragments upstream of mspA was measured and is indicated ...

To identify whether an additional promoter caused this increased expression of the lacZ gene, a 600-bp fragment ranging from −500 to −1100 with respect to mspA (“600 bp up”) was fused to lacZ. This upstream sequence had promoter activity as indicated by a β-galactosidase activity fivefold above background. However, the activities of both individual fragments of 500 bp and “600 bp up” were 11-fold below the activity of the complete 1,100-bp construct (Fig. (Fig.4B).4B). Thus, it was concluded that both fragments alone are not able to drive high expression of the lacZ gene. We further investigated the “600 bp up” fragment by dividing it into three 200-bp pieces. These 200-bp fragments (−500 to −700, −700 to −900, and −900 to −1100 upstream of mspA) were inserted upstream of the original 500-bp fragment of the mspA UTR, resulting in three additional 700-bp constructs (Fig. (Fig.4C).4C). The β-galactosidase activities of the original 700-bp and 1100-bp fragments were high and almost identical as observed earlier (Fig. (Fig.4A).4A). In contrast, the other three 700-bp constructs, consisting of the 200-bp fragments fused to the 500-bp promoter fragment via a restriction site, which added a 14-bp spacer between the two DNA fragments, only slightly increased the activity (Fig. (Fig.4C).4C). This included the 200-bp fragment immediately upstream of the 500-bp promoter fragment and indicated a dependence of the activation of PmspA on the phasing of the DNA helix.

Size and half-life of the mspA transcript.

To further characterize expression of the mspA gene in M. smegmatis, the size and half-life of the mspA transcript were determined by Northern blot experiments. An RNA probe that covered 355 bp of mspA from position +25 after the putative start codon to position +380 and included a T7 RNA polymerase promoter (see Table S4 in the supplemental material) was designed. First, the specificity of the probe for the mspA transcript was examined because M. smegmatis contains four similar msp genes that have 87% (mspB), 90% (mspC), and 74% (mspD) DNA sequence identity in the region covered by the mspA probe. To this end, RNA was prepared from wild-type M. smegmatis and from the ΔmspA mutant strain MN01 (42). A Northern blot did not reveal a transcript for the ΔmspA mutant, demonstrating that the RNA detected with the probe in wild-type M. smegmatis was indeed the mspA mRNA (Fig. (Fig.5A).5A). The length of the mspA transcript was determined to be approximately 900 bases by comparison to digoxigenin-labeled RNAs of known lengths. Considering the 636-bp length of the mspA gene and the 135-bp length of the 5′ UTR, it is concluded that the transcription of the mspA gene terminates 130 bp after the stop codon. However, no intrinsic terminator was found in this region.

FIG. 5.
Size and stability of the mspA mRNA. (A) Size of the mspA mRNA. RNA from either M. smegmatis SMR5 or MN01 was blotted and detected with digoxigenin-labeled RNA probes for mspA (top blot) and 16S rRNA (bottom blot). Lanes: 1, MN01; 2, SMR5. (B) Stability ...

To determine the half-lives of the mspA transcripts, RNA was isolated from wild-type M. smegmatis at different times after inhibition of transcription by rifampin. Northern hybridization was used to analyze the RNA (Fig. (Fig.5B).5B). The intensities of the mspA mRNA bands were quantified by image analysis, and the half-life of the mspA transcript was determined to be 6 minutes (Fig. (Fig.5C).5C). Surprisingly, the stability of the mspA transcript is dependent on the mspA expression construct. When the original promoter fragment of mspA was replaced by the constitutive mycobacterial promoters Pimyc and Psmyc (16), episomal expression decreased the half-lives of these mspA transcripts to 1 min and 2.3 min, respectively (Fig. (Fig.5D).5D). Thus, no mspA mRNA was detected after 5 min for the plasmid-based mspA expression cassettes in contrast to wild-type mspA transcripts, which were still detectable after 10 min (Fig. 5B and D). Faster RNA degradation was also observed on Northern blots with RNA isolated from strains containing mspA expression plasmids compared to the distinct bands for mspA mRNA from wild-type M. smegmatis (not shown).

Expression of mspA depends on the growth phase.

To examine whether transcription from the corresponding promoters was dependent on the growth phase of M. smegmatis, RNA was prepared from cells harvested at optical densities ranging from 0.1 to 3.0. Primer extension experiments revealed that transcription of mspA is maintained throughout early and exponential growth but decreased sharply after the cells entered stationary phase (Fig. (Fig.1C).1C). Quantitative image analysis showed that the amount of the shorter mspA mRNA was reduced 25-fold in stationary phase, indicating that mspA expression is dependent on the growth phase of M. smegmatis. To examine whether the weaker promoter using the TSP at position −153 had any regulatory function, we compared the amounts of the two transcripts in M. smegmatis grown in Middlebrook 7H9 medium under different conditions. Neither a shift in temperature from 37°C to 28°C or to 45°C nor an increased osmolarity (0.5 M NaCl) resulted in any detectable change of the ratio in the two mRNAs (data not shown).

Different environmental signals regulate transcription of the mspA gene.

To identify signals that regulate the expression of porin genes in M. smegmatis, we analyzed mspA expression under conditions that are known to modify porin gene expression in E. coli. Since nutrient limitation is among those signals (19), Hartmans-de Bont medium, which contains single compounds, such as carbon, nitrogen, and phosphorous sources, was used and reduced the amounts of glycerol, ammonium sulfate, and potassium phosphate to 11 mM, 150 μM, and 96 μM, respectively. These concentrations were shown to limit the growth of M. smegmatis in HdB medium significantly (data not shown), consistent with previous results (41). RNA was prepared from M. smegmatis grown at the indicated conditions. Both mspA mRNA and 16S rRNA were detected in the same blot using a mixture of specific probes (see Table S4 in the supplemental material). The intensities of the mspA mRNA bands were normalized to those of the corresponding 16S rRNA to eliminate differences in the total amount of RNA loaded on the gel. Northern blots with RNA from M. smegmatis revealed that mspA mRNA levels are decreased under carbon and phosphate limitation, while limitation of nitrogen elevated mspA mRNA levels (Fig. (Fig.6).6). The amount of mspA mRNA was unchanged at 28°C compared to 37°C but decreased more than 10-fold at 42°C. Increased osmolarity by addition of 0.5 M sodium chloride and 10% glucose decreased the amount of mspA mRNA 50- and 1.4-fold, respectively. Hydrogen peroxide (18 mM) decreased mspA mRNA levels fourfold, whereas no mspA mRNA was detected when M. smegmatis was exposed to 10% ethanol or when the pH of the medium was reduced to 3 (Fig. (Fig.6).6). Since pH values in soil are higher than pH 3 (14), the pH of the medium was varied from 4.5 to 6.8, which are physiologically more relevant pH values for M. smegmatis. Cells were grown in neutral HdB medium to an optical density of about 0.8, harvested, and resuspended in HdB medium with modified pH for 3 h. Then, RNA was isolated and analyzed by Northern hybridization with an mspA-specific probe. These experiments revealed that the amount of mspA mRNA declined with decreasing pH. At a pH of 4.5, no mspA transcripts were detectable (Fig. (Fig.7).7). Incubation of M. smegmatis at pH 4.5 for 3 h did not affect the viability of the cells compared to the control at pH 6.8, as determined by growth on Middlebrook 7H10 plates, in agreement with a previous report (34).

FIG. 6.
Dependence of mspA mRNA levels on growth conditions. (A) Northern blot analysis of RNA from M. smegmatis SMR5 and RNA probes specific for mspA transcripts and the 16S rRNA. co, growth in HdB medium at 37°C; C limitation, carbon limitation (11 ...
FIG. 7.
Dependence of mspA mRNA levels on the pH of the culture. (A) RNA was prepared from M. smegmatis SMR5 grown in modified HdB medium including 50 mM citrate buffer at pH 6.8, 5.5, 5.0, and 4.5. Detection of the RNA on positively charged nylon membranes using ...

The low mspA mRNA levels at pH 4.5 result from a specific regulatory event.

The yield of total RNA from M. smegmatis at pH 4.5 was significantly lower than at pH 6.8 (not shown). Therefore, we examined whether the very low levels of mspA mRNA at pH 4.5 were a consequence of generally reduced transcription in M. smegmatis under those conditions rather than being caused by a specific regulatory event. To this end, we chose sigA as a reference gene, which encodes the main σ-factor and is frequently used for quantification of mRNA levels in mycobacteria because it is transcribed constitutively under many conditions (22). Dot blot experiments with RNA isolated from M. smegmatis grown in HdB medium at pH 4.5, 5.5, and 6.8 showed that the amounts of sigA mRNA and 16S rRNA did not change under these conditions in contrast to the strongly reduced amount of mspA mRNA at pH 4.5 (Fig. (Fig.8A).8A). Further dot blot experiments demonstrated that transcription of plasmid-encoded mspA genes in the ΔmspA mutant M. smegmatis MN01 (42) did not change at low pH in the medium after replacement of the mspA promoter by the constitutive mycobacterial promoters Pwmyc, Pimyc, or Psmyc (16) (Fig. (Fig.8B).8B). In contrast, a significant reduction of mspA mRNA at pH 4.5 was observed for both wild-type M. smegmatis and the ΔmspA mutant containing a plasmid with the mspA gene under its own promoter (pPOR6 [see Table S1 in the supplemental material[). In addition, fusion of an 1,100-bp fragment containing the mspA promoter and its putative UTR to the E. coli lacZ gene conferred pH sensitivity to the lacZ mRNA levels (Fig. (Fig.8C).8C). This regulatory event was not observed by β-galactosidase activity measurements, presumably due to the already high β-galactosidase levels before transfer of M. smegmatis to the low-pH medium (not shown). In conclusion, these results (i) excluded the possibility that a general transcription defect at pH 4.5 causes the low mspA mRNA levels in M. smegmatis and (ii) demonstrated that at pH 4.5, either transcription from the mspA promoter is specifically repressed or that the 5′ UTR of the mRNA transcribed from the mspA promoter confers a pH-dependent instability to the corresponding mRNA.

FIG. 8.
mspA mRNA levels are specifically reduced at pH 4.5. Dot blot analysis was performed. (A) RNA was prepared from M. smegmatis SMR5 cultures grown at pH 6.8, 5.5, and 4.5 in HdB medium and was detected on dot blots using specific RNA probes for mspA mRNA, ...


Identification and activity of the mspA promoter.

Here, we present the first analysis of porin gene expression in mycobacteria. Primer extension analysis revealed a strong signal for a TSP at position −135 upstream of the mspA gene. Single point mutations identified the −10 sequence of the mspA promoter, which is similar to σA promoters of M. smegmatis (26). A potential −35 region was found 17 bp upstream of the −10 region (Fig. (Fig.2).2). However, we did not examine the importance of this element for activity of PmspA. The observation that the A2C mutation in the −10 region of PmspA completely eliminated the β-galactosidase activity of transcriptional fusions of the 1,100-bp DNA fragment upstream of mspA with lacZ (Fig. (Fig.3)3) demonstrates that there is no promoter in addition to PmspA. Surprisingly, a 500-bp fragment of the mspA 5′ region yielded only low expression of lacZ in a transcriptional fusion, whereas lacZ expression increased 12-fold with a 700-bp fragment. Longer fragments did not increase expression of lacZ further. Thus, it is concluded that approximately 700-bp DNA upstream of mspA are required for full activity of the PmspA promoter. Importantly, activity of a 700-bp DNA upstream of mspA was only twice as high as the basal activity of the 500-bp fragment with PmspA when the 200-bp activating element (−500 to −700) was separated by a 14-bp spacer from the 500-bp basal promoter (Fig. (Fig.4C).4C). Thus, activation of PmspA by the 200-bp element was dependent on the phasing of the DNA helix, an indicator of binding of an activator protein (13, 24). The requirement for rather long upstream regions for full activity has been observed for other mycobacterial promoters. For example, a region between 436 bp to 559 bp upstream of katG of Mycobacterium tuberculosis is required for full transcription in M. smegmatis, although no additional promoter was detected in this fragment. However, in this case the phasing between the UTR and the promoter was not critical for promoter activity (27). Another enhancer-like element was identified between 670 bp and 760 bp upstream of the mas gene of M. tuberculosis, but it is unknown whether proteins bind to this DNA (40). Further experiments are required to identify the putative activator protein for transcription of mspA.

The 5′ UTR contributes to the stability of the mspA transcript.

The mspA transcript has a defined size of approximately 900 bases (Fig. (Fig.5A).5A). This means that the mRNA ends approximately 130 bases after the mspA gene, considering the lengths of the gene (636 bp) and the 5′ UTR (135 bp). This is within the intergenic region between mspA and msmeg0956 (43) and indicates that the mspA gene is transcribed independently and is not part of an operon. A U trail following a hairpin structure is an essential component of intrinsic transcription terminators in other bacteria (47) but was not detected within this region. It was proposed that terminators of transcription in mycobacteria do not need a U trail when they have RNA hairpins with a stem length exceeding 27 bp (47). However, the 3′ end of the mspA transcript contains only sequences capable of forming hairpins with a stem length shorter than 5 bp. The half-life of the mspA transcript is 6 minutes during exponential growth at 37°C. For E. coli, an average messenger stability was reported to be 2.4 min at 37°C (37), whereas to our knowledge, nothing is known about mRNA stability in mycobacteria. An altered 5′ end in transcriptional fusions of mspA with other promoters increased mRNA degradation three- to sixfold (Fig. (Fig.5A),5A), confirming a pivotal role of the mspA 5′ UTR in stabilizing mspA transcripts. Secondary structures, such as stem-loops at the 3′ and 5′ ends of the transcript, often protect mRNA from degradation by ribonucleases (36). Analysis of the mspA 5′ UTR with the RNAStructure software (23) revealed a secondary structure at position −70 to −120 (Fig. (Fig.9),9), which may be involved in stabilizing the mspA transcript in a manner similar to that of the stem-loops at the 5′ end of the exceptionally stable ompA transcript of E. coli (12). In fact, the mspA hairpin is more stable (calculated ΔG = −13.4 kcal mol−1) than a modified ompA stem-loop with a stem length of 6 bp (calculated ΔG = −9.9 kcal mol−1), which was sufficient to increase the half-life of the ompA transcript in E. coli from 3.9 min to 20 min (2). An alternative explanation for the stability of the mspA mRNA might be protection from degradation by RNases upon binding of antisense RNA. Indeed, antiparallel transcripts were observed for the 5′ end of the mspA transcript (data not shown). However, experimental evidence for the role of antisense RNA in regulating the stability of the mspA mRNA is lacking.

FIG. 9.
Hairpin structure in the 5′ UTR of mspA. The picture was drawn according to secondary structure predictions of RNAStructure 4.3. The ΔG of the depicted loop forms was calculated to be −13.4 kcal/mol. The start codon of mspA and ...

Many environmental signals alter mspA expression.

Porins are important for nutrient uptake both in gram-negative bacteria (30) and in M. smegmatis (42, 43). In E. coli, the OM permeability is elevated by increasing the amount of the larger porin OmpF and decreasing the level of the smaller porin OmpC under conditions of nutrient limitation and vice versa under adverse conditions (33). Therefore, we compared regulation of expression of mspA with that of ompF, because MspA and OmpF are the main porins that determine OM permeability of M. smegmatis and E. coli, respectively, under many conditions. Glucose limitation leads to 20-fold-higher ompF mRNA levels compared to the level in medium containing excess glucose (20). In contrast, the amount of mspA mRNA was reduced threefold even after a slight reduction of the glycerol concentration from 22 mM to 11 mM and increased by 40% under nitrogen limitation (Fig. (Fig.6).6). Interestingly, M. smegmatis did not grow in HdB medium at glycerol concentrations lower than 11 mM in striking difference to E. coli, which grows with its maximal rate in a medium containing as little as 10 μM glucose (11). Thus, M. smegmatis does not only appear to experience C limitation at concentrations 1,000-fold higher than E. coli but also responds in an opposite manner by shutting down porin synthesis. Phosphate limitation had the largest effect and caused an almost complete loss of mspA mRNA. In E. coli, expression of another porin, PhoE, with specificity for anions is induced under low phosphate conditions (35), while to our knowledge, the effects of these conditions on expression of general porins such as OmpF have not been examined. Thus, M. smegmatis may induce expression of an unknown phosphate-specific porin and reduce expression of MspA, which does not appear to be well suited for diffusion of anions through the highly negatively charged constriction zone (10). However, alternative explanations such as a transition of M. smegmatis to a state of low metabolic activity under unfavorable conditions cannot be excluded.

As M. smegmatis enters stationary phase, mspA mRNA is barely detectable anymore (Fig. (Fig.1C).1C). This is similar to the reduced synthesis of OmpF by E. coli in response to depleted nutrient sources (33). The amount of ompF mRNA in E. coli decreases threefold during growth at temperatures of 37°C and above compared to growth at 24°C (1). Physiologically, the reduced expression of ompF makes sense because diffusion of small solutes through water-filled channels is accelerated at higher temperatures. A similar effect was observed for M. smegmatis. mspA mRNA levels were reduced fivefold at 42°C compared to those at 37°C (Fig. (Fig.6).6). The osmolarity of the medium is high in the intestine, the natural environment of E. coli. Under these conditions OmpC is the predominant protein and ompF expression is repressed (33). Similarly, both 0.56 M glucose (10%) and 0.5 M NaCl reduce mspA mRNA levels in M. smegmatis. Oxidative stress activates the micF promoter via SoxS and therefore represses ompF expression in E. coli (33). A similar effect was observed for M. smegmatis, which reduced mspA mRNA levels by threefold in the presence of 18 mM H2O2. M. smegmatis responds to the presence of another toxic agent, ethanol, by down-regulation of porin gene expression (Fig. (Fig.6)6) in a manner similar to that of E. coli (33). The mspA mRNA gradually declined with decreasing pH and was completely absent at pH 4.5 (Fig. (Fig.7).7). It may be argued that pH 4.5 is not physiologically relevant for M. smegmatis. However, M. smegmatis is able to maintain an intracellular pH of 6.1 to 7.2 during growth at an extracellular pH of 4.5 (34) and is highly abundant in brook sediments and forest soils at pH 3.5 to 4.3 (14, 15). Repression of ompF expression as a result of low pH was also reported for E. coli (7, 18, 39, 44, 46). A progressive reduction of the OM permeability over a range of pH 6.8 to 4 would reduce proton influx and support an adaptation of M. smegmatis to acidic habitats (32). The mechanism by which M. smegmatis reduced mspA mRNA levels at low pH is specific and is mediated by the 5′ UTR of the mspA gene as demonstrated by the unchanged levels of three mspA transcripts with different UTRs at pH 4.5 (Fig. (Fig.8B8B).

In conclusion, we have identified environmental signals that alter porin gene expression in M. smegmatis. The results show that M. smegmatis protects itself by reducing the OM permeability via down-regulation of porin gene expression similar to the response of E. coli to toxic agents. This is indicative of a general protection mechanism of bacteria with a second membrane and probably holds true for other mycobacteria as well. The response of porin gene expression to nutrient limitation by M. smegmatis is different from that by E. coli and reflects the fact that these bacteria have evolved different metabolisms to thrive in their habitats. We further discovered that mspA transcripts are stabilized by their original 5′ UTR, probably due to the formation of protective secondary structures, and that an enhancing fragment between 500 and 700 bp upstream of mspA is required to fully activate PmspA. Thus, the regulation of mspA expression by environmental signals includes both transcriptional and posttranscriptional mechanisms.

Supplementary Material

[Supplemental material]


We thank Chuck Turnbough for invaluable advice; Sabine Ehrt for the pMlacZsd vector; and Chuck Turnbough, Jason Huff, and Olga Danilchanka for critically reading the manuscript. Preliminary sequence data for Mycobacterium smegmatis were obtained from The Institute for Genomic Research website at http://www.tigr.org.

This work was supported by the Deutsche Forschungsgemeinschaft by grants to M.N. (NI 412) and fellowships to D.H. (Graduiertenkolleg 805) and I.E. (Graduiertenkolleg 40).


[down-pointing small open triangle]Published ahead of print on 1 December 2006.

Supplemental material for this article may be found at http://jb.asm.org/.


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